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Sequencing of Tryptic Peptides Using Chemically Assisted Fragmentation and MALDI-PSD

  • John Flensburg
  • Maria Liminga
Protocol
  • 2.2k Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become the preferred method for high-throughput identification of proteins using peptide mass fingerprinting (PMF), due to its ease of automation, short analysis time, relatively high tolerance towards contaminants, high sensitivity, and mass accuracy (1). In this technique, proteins are typically separated by twodimensional (2-D) gel electrophoresis, enzymatically in-gel digested with trypsin, extracted from the gel, and analyzed by MALDI-TOF MS. The resulting peptide mass fingerprints are compared to theoretical fingerprints from a protein- or DNA-sequence database for identification. In comparison to other ionization techniques such as electrospray ionization (ESI), the soft ionization induced by MALDI predominantly generates singly charged ions, which allows for a relatively easy interpretation of acquired spectra. Unfortunately, identification is not always unambiguous for a substantial fraction of the peptides analyzed, and it is not unusual that only a few peptides are recovered from an in-gel digest, especially when the protein is poorly expressed. To further improve the protein identification rate, amino acid sequence information from tryptic peptides is necessary. However, it is a well known fact that direct sequencing using MALDI post-source decay (PSD) often results in poor and unpredictable fragmentation patterns, which are mostly impossible to interpret (2). Singly charged tryptic peptides, formed during MALDI ionization, do not fragment readily because there is not enough internal energy available to move the ionizing proton from the basic C-terminal to the peptide backbone to induce fragmentation. In ESI, this problem is easily avoided by selecting doubly protonated peptides, which fragment readily.

Keywords

Tryptic Peptide Peptide Mass Fingerprinting Unambiguous Identification Sulfonation Reaction Amino Acid Sequence Information 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Shevchenko, A., Jensen, O. N., Podtelejnokov, A., et al. (1996) Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc. Natl. Acad. Sci. USA 93, 1440–1445.CrossRefGoogle Scholar
  2. 2.
    Spengler, B. (1997) Post-source decay analysis in matrix-assisted laser desorption/ionization mass spectrometry of biomolecules. J. Mass Spectrom. 32, 1019–1036.CrossRefGoogle Scholar
  3. 3.
    Keough, T., Lacey, M. P., and Youngquist, R. S. (1999) A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry. Proc. Natl. Acad. Sci. USA 96, 7131–7136.PubMedCrossRefGoogle Scholar
  4. 4.
    Liminga, M., Carlsson, U., Larsson, C., et al. (2001) New water stable chemistry for improved amino acid sequencing by derivatization postsource-decay (dPSD) using Ettan MALDI-TOF with a quadratic field reflection. Proc. 49th ASMS Conf. Mass Spectrometry and Allied Topics, Chicago, IL.Google Scholar
  5. 5.
    Keough, T., Lacey, M. P., and Youngquist, R. S. (2002) Solid-phase derivatization of tryptic peptides for rapid protein identification by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 16, 1003–1015.PubMedCrossRefGoogle Scholar
  6. 6.
    Hellman, U. and Bhikhabhai, R. (2002) Easy amino acid sequencing of sulfonated peptides using post-source decay on a matrix-assisted laser desorption/ionization time-of-flight spectrometer equipped with a variable voltage reflector. Rapid Commun. Mass Spectrom. 16, 1851–1859.PubMedCrossRefGoogle Scholar
  7. 7.
    Flensburg, J. and Belew, M. (2003) Characterization of recombinant human serum albumin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. J. Chromatogr. A 1009, 111–117.PubMedCrossRefGoogle Scholar
  8. 8.
    Eklund, P., Andersson, H. O., Kamali-Moghaddam, M., Sundstr/:om, L., and Flensburg, J. (2003) Purification and partial characterization by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the recombinant transposase, TniA. J. Chromatogr. A 1009, 179–188.PubMedCrossRefGoogle Scholar
  9. 9.
    Keough, T., Lacey, M. P., and Youngquist, R. S. (2000) Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 14, 2348–2356.PubMedCrossRefGoogle Scholar
  10. 10.
    Wysocki, V. H., Tsaprailis, G., Smith, L. L., and Breci, L. A. (2000) Mobile and localized protons: a framework for understanding peppide dissociation. J. Mass Spectrom. 35, 1399–1406.PubMedCrossRefGoogle Scholar
  11. 11.
    Flensburg, J., Haid, D., Blomberg, J., Bielawski, J., and Ivansson, D. (2004) Applications and performance of a MALDI-TOF mass spectrometer with quadratic field reflectron technology. J. Biochemical and Biophysical Methods 60, 319–334.CrossRefGoogle Scholar
  12. 12.
    Anderson, U. N., Colburn, A. W., Makarov, A. A., et al. (1998) In-series combination of a magnetic-sector mass spectrometer with a time-of-flight quadratic-field ion mirror. Rev. Sci. Instrum. 69, 1650–1660.CrossRefGoogle Scholar
  13. 13.
    Zhang, W. and Chait, B. T. (2000) ProFound: An expert system for protein identification using mass spectrometric peppide mapping information. Anal. Chem. 72, 2482–2489.PubMedCrossRefGoogle Scholar
  14. 14.
    Field, H. I., Fenyö, D., and Beavis, R. C. (2002) RADARS, a bioinformatics solution that automates proteome mass spectral analysis, oppimises protein identification, and archives data in a relational database. Proteomics 2, 36–47.PubMedCrossRefGoogle Scholar
  15. 15.
    Bell, P. J. L. and Karuso, P. (2003) Epicocconone, a novel fluorescent compound from the fungus Epicoccum nigrum. J. Am. Chem. Soc. 125, 9304–9305.CrossRefGoogle Scholar
  16. 16.
    Mackintosh, J. A., Choi, H., Bae, S., et al. (2003) A fluorescent natural product for ultra sensitive detection of proteins in 1-D and 2-D gel electrophoresis. Proteomics 3, 2273–2288.PubMedCrossRefGoogle Scholar
  17. 17.
    The Arabidopsis Genome Initiative. (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815.Google Scholar
  18. 17.
    The Arabidopsis Genome Initiative. (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815.CrossRefGoogle Scholar
  19. 18.
    Aebersold, R. and Goodlett, D. R. (2001) Mass spectrometry in proteomics. Chem. Rev. 101, 269–295.PubMedCrossRefGoogle Scholar
  20. 19.
    Hoffmann, R., Metzger, S., Spengler, B., and Otvos, L. (1999) Sequencing of peppides phosphorylated on serines and threonines by post-source decay in matrix-assisted laser desorppion/ionization time-of-flight mass spectrometry. J. Mass Spectrom. 34, 1195–1204.PubMedCrossRefGoogle Scholar
  21. 20.
    Metzger, S. and Hoffmann, R. (2000) Studies on the dephosphorylation of phosphotyrosine-containing peppides during post-source decay in matrix-assisted laser desorppion/ ionization. J. Mass Spectrom. 35, 1165–1177.PubMedCrossRefGoogle Scholar
  22. 21.
    Qin, J. and Chait, B. T. (1997) Identification and characterization of post translational modification of proteins by MALDI ion trap mass spectroscopy. Anal. Chem. 69, 4002–4009.PubMedCrossRefGoogle Scholar
  23. 22.
    Bhikhabhai, R., Algotsson, M., Carlsson, U., et al. (2004) Amino acid sequencing of sulfonic acid-labeled tryptic peptides using post-source decay and quadratic field MALDIToF mass spectrometry. In Kamp, R. M., Calvete, J. J., and Choli-Papadopoulou, T. (eds.), Principles and Practice, Methods in Proteome and Protein Analysis Springer-Verlag, Berlin, Heidelberg, pp. 279–296.Google Scholar
  24. 23.
    Keough, T., Youngquist, R. S., and Lacey, M. P. (2003) Sulfonic acid derivatives for peppide sequencing by MALDI MS. Anal. Chem. 75(7), 156A–165A.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • John Flensburg
    • 1
  • Maria Liminga
    • 1
  1. 1.Amersham Biosciences AB, GE HealthcareUppsalaSweden

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